April 5, 1955 NOTES 1911

cooled, diluted with ether and extracted with dilute hydro- chloric acid. The ether solution was washed with water, dried and concentrated. 4ddition of ethanol gave 19.6 g. of product, m.p. 101-103.5 . This was recrystallized from ethanol a;d gave 18.7 g. (73%) of yellow product, m.p. 102-104.5 . Chromatography on alumina gave a colorless product, 15.4 g., m.p. 104.4-105.5", [ a I z 6 D -37.1' (c 1.24, chloroform, 1 dm.).

Anal. Calcd. for Ca2H4104: C, 78.01; H, 9.00. Found: C, 78.13; H , 9.13.

3,9-Acetoxy-A~-norcholenic Acid .-A suspension of 4.93 g. (0.01 mole) of benzyl 3~-acetoxy-A6-norcholenate in 200 ml. of 95% ethanol was shaken with pre-reduced palladium (from 200 mg. of oxide) under hydrogen. At the end of 13 hr. the uptake of hydrogen ceased a t 0.01 mole. The solu- tion was filtered and evaporated to dryness in OQCUO. The crude product, m.p. 197.5-199', was recrystallized from acetone-petroleum ether to give 3.7 g., m.p. 197.5-198.5', [aIz6D -42' (c 1.14, chloroform, 1 dm.).

Anal. Calcd. for C26Hs804: C, 74.59; H , 9.52: neut. equiv., 402.6. Found: C, 74.44; H, 9.37; neut. equiv., 405,409. DEPARTMENT OF CHEMISTRY YALE UNIVERSITY NEW HAVEN, CONN.

Studies on the Chemistry of Heterocyclics. XXVZI.' a,@-Acetylenic Acids and their Esters in

the Thiophene Series BY A. J. OSBAHR,~ A. VAITIEKUNAS AND F. F. NORD

RECEIVED OCTOBER 15, 1954

Continuing our studies on acetylenic derivatives in the thiophene series3** we report in this paper the preparation of @acetylenic acids and their esters. The intermediates (thienylacetylenes) were prepared according to the earlier procedure de- veloped in this Laboratory.6 We were able to iin- prove the yields reported earlier. The yield for 2- thienylacetylene could be increased up to 80% while that for 2,5-dichloro-3-thienylacetylene amounted to 91%. The yield of 5-chloro-2- thienylacetylene was, however, low (28%).

The direct carbonation of the sodium salts of thienylacetylenes did not lead to any of the de- sued a,@-acetylenic acids. We observed that neither pressure nor different solvents had any effect on the products of the reaction. In all cases small amounts of 2-thenoic acids were isolated only. This phenomenon was observed earlier while studying y-hydroxy-a,@-acetylenic acids. Perhaps the acetylenic acids once formed were hydrated to the keto acids. The latter on cleavage furnished the corresponding thenoic acids.

An alternate procedure via the a,@-acetylenic esters furnished the desired a,O-acetylenic acids in this series in satisfactory yields. The sodium salts of the thienylacetylenes were prepared by applying sodium amide in liquid ammonia as reported earlier.4 The latter were treated with ethyl chloro- carbonate giving the corresponding esters as

(1) For a comprehensive review of some recent developments in the field of thiophene see F. P. Nord, A. Vaitiekunas and L. J. Owen, Forlschritte d . chcm. Forschung, S, 309 (1955).

(2) Condensed from a portion of the thesis of A. J. 0. submitted to the Graduate School of Fordham University in fulfillment of the re- quirements of the M.Sc. degree.

(3) A. Vaitiekunas and F. F. Nord, Tars JOURNAL, 76, 2733 (1954). (4) A. Vaitiekunas and F. F. Nord, ibid., 76, 2737 (1954). (5) A. Vaitiekunas and F. F. Nord, J . Org. Chem., 19, 902 (1954).

NaNH2

"a RC=CH -

ClCOOCzHb R C z C . Na - RCECCOOCIH~

Ether

The previously applied method for the preparation of y-hydroxy-@acetylenic esters in this series was modified by adding a large excess of ethyl or methyl chlorocarbonate to the reaction mixture and refluxing i t for several hours. Complete separation of the acetylenic esters from the ac- companying acetylenes by distillation was diffi- cult, but separation could be effected by precipita- tion of the acetylene as the copper salt followed by distillation of the ester in vacuo.

The properties and yields of the a,B-acetylenic esters are recorded in Table I.

TABLE I PROPERTIES AND YIELDS OF THE THIENYLPROPIOLIC ESTERS

B.P. (1 Analyses, %

Ponxid Ethyl mm.), Yfefd, Calcd. proplolate O C . % C H C1 C H cf

2-Thtenyl- 95-98 91 60 4 . 4 5 , , . 5 9 . 8 4 . 2 .. 6-Chloro-2-

thienyl 56-58 45 50.35 3.26 16 .5 50.5 3 . 5 17 2,5-Dichloro-3-

thienyl 92-96 80 43.37 2 .41 28.59 43 .5 2 . 3 28 .3 3-Methyl-2-

thienyl 66-67 70 63 5 6 .29 . l . 6 3 . 8 5 . 4 . . While applying our earlier procedure for the

isolation of the free y-hydroxy-a,B-acetylenic acids4 the free 2- and 3-thienyl-a,B-acetylenic acids were obtained in satisfactory yields according to the reaction

(OH-) RCEC-COOC~H~ _L)

R-C=C-COOH + CzHaOH

The properties and yield of the a,O-acetylenic acids are recorded in Table 11.

TABLE I1 PROPERTIES AND YIELDS OF THE THIENYLPROPIOLIC ACIDS

Analyses, % Propiolic M.p., Yield, Calcd. Found

acid OC. yo C H C1 C H C1 2-Thienyl- 130-133 85 55.26 2 .63 . . . 5 5 . 0 2 . 8 . . 2,5-Dichloro-

5-Chloro-2-

3-Methyl-2-

3-thienyl 139-141 79 3 8 . 0 0.905 32.12 3 7 . 9 1 . 2 32 .0

thienyl 118-120 45 45.04 1 . 6 19.02 4 4 . 8 2 . 0 18 .9

thienyl 115-118 60 57 .8 3.61 . . . 5 6 . 0 3 . 8 . . Polymerization took place to a greater or lesser

extent in all the reactions carried out in this series. In the case of the 5-chloro compound the poly- merization was increased so far that only small yields of the desired products could be obtained. The polymerization of the acetylenic compounds in this series probably is due to the mobility of the electrons of the hetero atom.

1912 NOTES Vol. 77

Experimental Materials.-The thiophene used in this work was ob-

tained through the courtesy of the Monsanto Chemical Co., Inc., St. Louis, Mo. The 2-chlorothiophene and 2,5-di- chlorothiophene were obtained through the courtesy of the Jefferson Chemical Co., Inc., New York, N.Y. Ethyl chlorocarbonate, 3-methylthiophene and anhydrous am- monia were commercial products.

General Procedure for the Preparation of Ethyl Esters of Thienyl-a,p-acetylenic Acids.-To freshly prepared sodium amide (0.05 mole) in 350 ml. of liquid ammonia in a three- necked Bask there was added 0.05 mole of the thienylacetyl- ene or its derivatives dissolved in a threefold volume of ab- solute ether. After stirring for an additional one-half hour, the liquid ammonia was allowed to evaporate on a steam- bath, while replacing i t with absolute ether. The contents of the flask were cooled to 0-5’ and 0.3 of a mole of ethyl chlorocarbonate dissolved in 35 ml. of absolute ether was added slowly. The reaction mixture turned from red- brown to pale yellow. I t was allowed to reach room tem- perature and was stirred for an additional four hours. The sodium chloride was filtered off, the filtrate washed with 5% sodium carbonate solution, finally twice with ice-water, dried over anhydrous sodium sulfate and rectified.

General Procedure for the Hydrolysis of Ethyl Ester of Thienylw,p-acetylenic Acids.-One gram of ethyl ester of thienyl-a,&acetylenic acid dissolved in 15 ml. of benzene was added to a solution of 25 ml. of 1.5 N sodium hydroxide and the reaction mixture shaken a t room temperature for 48 hours. The isolation of the acid was carried out in the usual manner. The yields and elemental analyses of a,p- acetylenic acids are recorded in Table 11.

Attempts to Prepare the Thienyl-a,B-acetylenic Acids via the Direct Carbonation of the Sodium Salts of Thienylacetyl- ems.-Sodium acetylide was formed in the usual manner using 0.2 g. of iron nitrate plus 0.42 g. of sodium and 200 ml. of liquid ammonia. Ten grams of pure 2-thienylacetyl- ene was added dropmise over a period of one hour. The solution was then stirred ten minutes longer. The three- necked flask was then placed on a steam-bath and the am- monia driven off, using a stirrer and reflux condenser. Then 40 nil. of dry benzene were added from a dropping funnel to be used as solvent in the carbonation reaction. This solution containing the solvent and the sodium acetyl- ide salt was placed in a bomb and carbonated with 200 g. of Dry Ice for 24 hours. The reaction product was acidi- fied with 5 N sulfuric acid a t 0” and twice extracted with ethyl ether. The combined organic layers were extracted again with 10% sodium carbonate and the aqueous layer cooled to 0” and acidified with 3 N sulfuric acid giving the free acid. The product, if recrystallized from carbon tetra- chloride, was identified as 2-thenoic acid only.

Acknowledgments.-This study was aided in part by a grant from the Office of Naval Research. The analyses were carried out by Drs. G. Weiler and F. B. Strauss, Microanalytical Laboratory, Oxford, England. CONTRIBUTION KO. 295 DEPARTMENT OF ORGANIC CHEMISTRY AXD ENZYMOLOGY FORDHAM UVIVERSITY NEW YORK 58, N. Y.

Evidence from Titration Curves on the “Acyl Shift” in Proteins

BY CHARLES TANFORD RECEIVED OCTOBER 22, 1954

A recent paper by Lumry and Eyringl has drawn attention to the “acyl shift” which may occur a t peptide bonds adjacent to serine or threonine residues in proteins. This intramolecu- lar rearrangement appears to explain satisfac- torily the lability, during acid hydrolysis, of peptide bonds adjacent to serine or threonine residues. Lumry and Eyring make the further

( 1 ) R. Lumry and H. Eyring, J . Phys. Chcm., 68, 110 (1954).

suggestion that the acyl shift may be important under conditions much milder than those of acid hydrolysis and that, therefore, “acid titration ex- periments will almost universally need re-evaluation in light of this finding.”

The purpose of this note is to show that such re- evaluation of titration data is not necessary. In fact, titration data show that the acyl shift does not occur, a t least in proteins so far carefully examined, in the relatively mild acidities (down to pH 2) reached in titration studies.

The connection between the acyl shift and titra- tion curves lies in the fact that an amino group is released during the rearrangement.‘ Each serine or threonine residue capable of undergoing the acyl shift would therefore be expected to con- tribute a cationic group to the protein molecule. The total number of cationic groups would then be considerably greater than the number calculated from amino acid analysis on the basis that only arginine, lysine, histidine and terminal a-amino residues contribute to the number of cationic groups.

The total number of cationic groups of a protein molecule, present at the acid end-point of a titra- tion curve, is equivalent to the number of hydrogen ions bound in going from the isoionic point to the acid end-point.2 This number can therefore be accurately determined for those proteins for which the isoionic point can be established. For proteins soluble in water a t the isoionic point, this point may be established by direct measurement on de- ionized solutions. In some other proteins, where salt binding is not important, i t may be established as the point of intersection of titration curves a t different ionic strengths.

The pertinent data are available for five dif- ferent proteins and the total numbers of cationic groups in these proteins, as determined from titra- tion, are listed in Table I. The figures are com- pared with analytical data. It is seen that in every case the agreement between analytical and titra- tion data is within experimental The titrstion curves do not show any excess cationic groups due to acyl shift. If all serine and threo- nine residues were capable of undergoing an acyl shift alkaline to p H 2, the number of excess cationic groups would have been 26 for ribonuclease, 16 for lysozyme, 32 for 0-lactoglobulin, 50 for ovalbumin, and 46 for serum albumin.

To the five proteins of Table I should be added insulin.? In this protein the isoionic point has

(2) E. J. Cohn and J. T . Edsall, “Proteins, Amino Acids and Pep- tides,” Reinhold Publishing Corp., New York, N . Y., 1943, p. 446.

(3) For the sake of uniformity, all analytical data have been taken from the same compilation (Tristram, ref. 4 ) . To indicate the prob- able error involved one may examine the data for lysozyme. Here Tristram’s compilation gives the analysis of Lewis, e1 al., (ref. 5). Another analysis by Fromageot and de Garilhe (ref. 6) would lead to a total of 19.8 rather than 17.8 cationic groups. The average of these would correspond exactly to the number found by titration.

(4) G. R. Tristram in H. Neurath and K. Bailey, ed., “The Pro- teins,’’ Vol. 1. Part A, Academic Press, Inc., New York. N. Y., 1953, p. 181.

(5) J. C. Lewis, N. S. Snell, D. J. Hirschmann and H. Fraenkel- Conrat. J. Biol. Chcm., 186, 23 (1950). (6) C. Fromageot and M. P. de Garilhe, Biochim. Biophys. Acta, 4 ,

509 (1950). (7) C. Tanford and J. Epstein, THIS JOURNAL. ‘76. 2163 (1954).